development and evaluation of liquid crystal systems of...

© 2019 Journal of Pharmacy & Pharmacognosy Research, 7 (6), 441-453, 2019 ISSN 0719-4250

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Original Article | Artículo Original

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Development and evaluation of liquid crystal systems of combination of 5-fluorouracil and curcumin for cervical cancer cell line

[Desarrollo y evaluación de sistemas de cristal líquido de una combinación de 5-fluorouracilo y curcumina en una línea celular de cáncer cervical]

Sheba R David1, Syahira Abdul Refai2, Koh Rhun Yian2, Chun-Wai Mai2, Sanjoy Kumar Das3, Rajan Rajabalaya1*

1PAPRSB Institute of Health Sciences, Universiti Brunei Darussalam, Jalan Tungku Link BE1410, Bandar Seri Begawan, Brunei Darussalam. 2School of Pharmacy, International Medical University, No. 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala Lumpur, Malaysia.

3Institute of Pharmacy, Jalpaiguri, Pin 735101, West Bengal, India. *E-mail: [email protected]

Abstract Resumen

Context: Liquid crystalline gel, self-assembled vesicular systems covered with nonionic surfactants, are promising for use in drug delivery.

Aims: To develop and evaluate the liquid crystal (LC) systems of a combination of 5-fluorouracil and curcumin for a cervical cancer cell line.

Methods: The LC was formulated using water, surfactant, and glycerin. Optimization was carried out by using different surfactants. The formulations were chosen for various studies, such as pH determination, environmental scanning electron microscopy (eSEM), Fourier transform infrared spectroscopy (FTIR) and dissolution study. The synergism of the combination of 5-FU and curcumin on cervical cancer HeLa cell line was then analyzed using CompuSyn software.

Results: The formulations with 60% surfactant, the average particle size was in the ranges from 15.68 – 26.35 nm whereas with 40% surfactant, were 11.27 - 21.35 nm. The percentage of dissolution of 5-FU at pH 7 (PBS) A2: 40.23 > B2: 47.39 > X2: 50.36 > Y2: 56.36 compared to at pH 4 (vaginal simulated fluid, VSF) were A2: 36.23 > B2: 43.64 > X2: 45.67 > Y2: 49.01. The percentage of dissolution of curcumin of different formulations were at pH 7 (PBS) A2: 44.85 > B2: 51.36 > X2: 58.61 > Y2: 64.38 compared to at pH 4 (VSF) were A2: 41.03 > B2: 49.37 > X2: 57.85 > Y2: 68.75.

Conclusions: The combination of the drugs showed that there was a synergistic effect when it was being administered together. The combination of drugs in LC system had a sustained release as well as it was effective against cervical cancer HeLa cell line.

Contexto: El gel cristalino líquido, los sistemas vesiculares autoensamblados cubiertos con tensioactivos no iónicos, son prometedores para su uso en la administración de fármacos.

Objetivos: Desarrollar y evaluar los sistemas de cristal líquido (LC) de una combinación de 5-fluorouracilo y curcumina para una línea celular de cáncer cervical.

Métodos: El LC se formuló usando agua, tensioactivo y glicerina. La optimización se llevó a cabo utilizando diferentes tensioactivos. Las formulaciones se eligieron para diversos estudios, como la determinación del pH, la microscopía electrónica de barrido ambiental (eSEM), la espectroscopía infrarroja por transformada de Fourier (FTIR) y el estudio de disolución. Luego se analizó la sinergia de la combinación de 5-FU y curcumina en la línea celular HeLa de cáncer de cuello uterino utilizando el software CompuSyn.

Resultados: Para las formulaciones con 60% de tensioactivo, el tamaño de partícula promedio estuvo en el rango de 15,68 – 26,35 nm mientras que con 40% de tensioactivo, fueron 11,27 – 21,35 nm. El porcentaje de di-solución de 5-FU a pH 7 (PBS) A2: 40,23 > B2: 47,39 > X2: 50,36 > Y2: 56,36 en comparación con pH 4 (fluido simulado vaginal, VSF) fueron A2: 36,23 > B2: 43,64 > X2: 45,67 > Y2: 49,01. El porcentaje de disolución de curcumina de diferentes formulaciones fue a pH 7 (PBS) A2: 44,85 > B2: 51,36 > X2: 58,61 > Y2: 64,38 en comparación con pH 4 (VSF) fueron A2: 41,03 > B2: 49,37 > X2: 57,85 > Y2: 68,75.

Conclusiones: La combinación de los compuestos mostró que había un efecto sinérgico cuando se administraba en conjunto. La combinación de fármacos en el sistema LC tuvo una liberación sostenida y fue eficaz contra la línea celular HeLa del cáncer de cuello uterino.

Keywords: cervical cancer; curcumin; 5-fluorouracil; HeLa cell line; liquid crystal.

Palabras Clave: cáncer de cuello uterino; cristal líquido; curcumina; 5-fluorouracilo; línea celular HeLa.

ARTICLE INFO Received: May 12, 2019. Received in revised form: August 28, 2019. Accepted: September 4, 2019. Available Online: October 5, 2019.

Declaration of interests: The authors declare no conflict of interest. Funding: This study was supported and funded by School of Pharmacy, International Medical University, Kuala Lumpur, Malaysia [grant BPharm B01/09-Res (17)2012].


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David et al. Liquid crystal system for cervical cancer

http://jppres.com/jppres J Pharm Pharmacogn Res (2019) 7(6): 442

INTRODUCTION

Cervical cancer is defined as an abnormal cell growth affecting the cells lining the cervix (Cohen et al., 2019). Both the National Institute of Health and the National Cancer Registry, Malaysia, stated that cervical cancer is the third leading cancer among women in Malaysia and worldwide in the years of 2008 and 2006 (Ministry of Health Malay-sia, 2006). There are two types of cells lining the cervix, squamous cells and glandular cells and most cervical cancers are usually squamous cell carcinoma (Cohen et al., 2019). The main cause of cervical cancer is the human papilloma virus, which can be detected using the Pap smear test (Cohen et al., 2019).

Liquid crystal is a substance that possesses the properties of both liquid and solid where it may flow like water but the molecular structure of liq-uid crystal represents a crystal (Rajabalaya et al., 2017). Liquid crystal system is suitable to be used locally since the system has a bio-adhesion and penetration enhancement property thus the ab-sorption of drugs is faster (Bei et al., 2009; Ra-jabalaya et al., 2017). Due to the large internal sur-face area and honeycombed structure of the liquid crystal system, it allows high drug loading and enables incorporation of drugs with different physicochemical properties, hydrophilic, lipophilic and amphiphilic substances other than having a property of a sustained release drug delivery sys-tem (Rajabalaya et al., 2017). Curcumin or diferu-loylmethane is an active ingredient extracted from the rhizomes of Curcuma longa L. (family Zingibera-ceae) or also called as turmeric (Tuyaerts et al., 2019). Traditionally, it has been used as a spice in cooking and also used to treat illnesses such as infection of the bile duct and gall bladder as well as other inflammatory diseases (Tamvakopoulos et al., 2007). Curcumin has also been shown to have anti-oxidant, anti-proliferative and anti-inflammatory properties (Tamvakopoulos et al., 2007).

5-Fluorouracil (5-FU) is a widely known anti-metabolite that has been used in the treatment of cancer for many years. 5-FU competes with natural

substrates for thymidylate synthetase; thus, it is incorporated into RNA or DNA as false substrate. In the treatment of cervical cancer, 5-FU is usually given together with cisplatin or paclitaxel. The combination of curcumin and 5-FU has been stud-ied widely. Some studies showed that there is a synergistic effect between curcumin and 5-FU (Du et al., 2006; Ahn et al., 2010; Liu et al., 2017). The objectives of this research were to determine the suitable solvents and adjuvants for liquid crystal systems and formulation, to study the physico-chemical properties of the liquid crystal systems and the formulation, to study the dissolution of the drug and to the analyze the efficacy of the formulation on a cervical cancer cell line.

MATERIAL AND METHODS

Materials

Curcumin was purchased from Pi Chemicals Ltd. (Shanghai, China). The surfactant Polysorbate 80 (Tween 80) was acquired from EMD Millipore Corporation, Billerica, MA, USA. Glycerol, phos-phate buffer saline (PBS) tablets, Dulbecco’s modi-fied Eagle’s medium (DMEM), and bovine serum albumin were acquired from Sigma-Aldrich, St Louis, MO, USA. Caprylocaproyl macrogol-8-glyceride (Labrasol®) was kindly provided from Gattefossé (St Priest, France) and polyoxyl 35 cas-tor oil (Cremophor® EL) from BASF (Ludwigsha-fen, Germany). All other chemicals used were of analytical grade.

Curcumin and 5-FU combination liquid crystal preparation and optimization

Formulations of the liquid crystal were pre-pared based on studies carried out by Rajabalaya et al. (2017) and Das et al. (2018). Tween 80 was used as the surfactant while glycerin and water were used as the aqueous phase. Curcumin, which is lipophilic in nature, was mixed with Tween 80 and melted in water bath with constant sonication for 15 minutes. On the other hand, 5-FU, which is hydrophilic, was dissolved in water first, before adding to glycerin. Both parts were then poured into a universal bottle and mixed thoroughly using




a vortex mixer (Rajabalaya et al., 2016). The formu-lation was optimized using three different surfac-tants, Tween 80, Labrasol and Cremophor with four different concentrations of surfactant 20, 40, 60 and 80% and using three different concentra-tions of drug, 0.1, 0.3 and 0.5%.The formulations that met the optimal physiochemical properties were used for further research.

Physiochemical investigation

Organoleptic properties

Organoleptic properties such as odor and color were determined by using the LC samples.

Homogeneity test

A 0.5 gm of a sample was taken and pressed be-tween the index finger and thumb to check on the consistency of the formulation.

pH determination

Accurately weighed amount (0.5 g) of the sam-ple formulation was dissolved in 15 mL distilled water (pH 7.0) and the pH was measured using a calibrated pH meter, Mettler Toledo, USA (Table 1).

Encapsulation efficiency

The LC formulations were centrifuged at 10,000 rpm at a temperature of 5°C for 60 min in two cy-cles using a cooling centrifuge (Eppendorf® Model 5810R, Eppendorf, Hamburg, Germany) to isolate the drug-carrying vesicles from the unentrapped drug. The supernatant was then removed and lysed with methanol. The sediment was filtered through a 0.45 µm nylon disk filter and the free drug amount in the supernatant was measured using High-Performance Liquid Chromatography (1200 HPLC series, Agilent Technologies, Santa Clara, CA, USA) (Musa et al., 2017). The percent-age encapsulation efficiencies were determined based on the following formula [1] (David et al., 2013) and are presented in Table 1.

% Encapsulation efficiency = Total drug content - drug content in supernatant

Total drug content × 100 1

[1]

Particle size determination

An aliquot of 0.1 g of LC formulation was dilut-ed with 5 mL double distilled water and sonicated for 30 seconds in an ice bath. Particle size of the sample was analyzed at a scattering angle of 173° at a temperature of 250°C (Rajabalaya et al., 2016). One mL of solution was injected into a zetasizer cuvette and scanned in Malvern Zetasizer Nano ZS (Malvern Instruments Limited, United King-dom) (Table 1).

Environmental Scanning Electron Microscopy (ESEM)

A LC gel weighing 0.1 g was placed on a hold-ing disk and a drop of distilled water was added. The disk was placed in a chamber inside an Envi-ronmental Scanning Electron Microscope (eSEM, Quanta 450 FEG; FEI, Hillsboro, OR, Fei Company, USA). Water within the sample was allowed to evaporate before examination. The sample formu-lation was placed in the sample holder and scanned (Rajabalaya et al., 2016).

Fourier Transform Infrared Spectroscopy (FTIR)

The IR spectra of the drugs, 5-FU crystal and powdered curcumin were measured individually and as combination by mixing both ingredients. The drugs combination was made into powder form before adding KBr. The thin film, formed by compressing the drug powder and KBr disc, was analyzed using Shimadzu FTIR-8400S FTIR spec-trometer at the wavelength of 500 – 4000 cm-1 (Ra-jabalaya et al., 2013).

Dissolution

Dissolution study was carried out to analyze the in vitro drug release profile of the drugs. Disso-lution study was carried out in modified USP dis-solution apparatus II (Weng and Parrott, 1983) for release studies of gels using two different simulat-ed conditions, in vaginal simulated fluid (VSF) and PBS. VSF was prepared with the method suggest-ed by Owen and Fatz (1999) The weighed amount (5 g) of gel was placed in 900 mL of vehicle (Weng and Parrott, 1983), VSF/PBS at 37 ± 0.5°C with paddle speed set to 50 rpm. The dissolution medi-um (900 mL) for gels and combination gels was




0.5% (m/v) solution of sodium lauryl sulphate in water (Owen and Katz, 1999). The 200 µL sample from the dissolution medium was taken every hour for 8 hours and replaced with their respective vehicle. The samples taken were made up to 10 mL and the absorbance were determined using dual beam UV-Visible spectrophotometer, PerkinElmer, USA, at wavelengths of 250 nm and 421 nm for 5-FU and curcumin, respectively.

Drug efficacy study

Cervical cancer (HeLa) cell line was used in this study. The cells were cultured in Dulbecco’s Modi-fied Eagle Medium (DMEM) with 10% Fetal Bo-vine Serum (FBS) and 1% penicillin in an incubator (RS Biotech Laboratory Equipment, UK) supplied with 5% carbon dioxide. The cells were then plated onto 96-well plate and treated with blank liquid crystal, curcumin alone, 5-FU alone, combination of both drugs or molecular grade dimethyl sulfox-ide (DMSO) as control. MTT (3-(4,5-dimethyl-thiazol-2-yl)-2-5-diphenyltetrazolium bromide), a yellow tetrazole, was then added to the plate. DMSO of analytical grade was then used to break down MTT into purple formazan, which stained the living cells. The viability of the cells was meas-ured using Opsys MRTM Microplate Reader (Dynex Technologies Inc., USA).

Statistical analysis

Statistical analyses of data were undertaken using SPSS v8.0 (IBM, Armonk, NY, USA). Analysis of variance and the paired t-test were applied. p<0.05 was considered significant. Values are expressed the mean ± standard deviation.

RESULTS

Curcumin and 5-FU combination liquid crystal preparation and optimization

The results of the formulations prepared with Tween 80, Labrasol and Cremophor for percentage of cytotoxicity of different surfactants on HeLa

cells have been tabulated in Table 2. In terms of toxicity, both Tween 80 and Labrasol did not elicit toxic effect on the cells unlike Cremophor at con-centrations above 3 µg/mL. Furthermore, the for-mulations with Labrasol did not follow the desired criteria for vaginal drug delivery. Thus, based on these results, Tween 80 was selected and used in further experiments. Table 1 shows the results for the various physiochemical parameters and the formulation composition of the formulations.

Physiochemical investigation

All liquid crystal formulations prepared with Polyoxyethylene (20) sorbitan monooleate, also known as Tween 80, were light yellow in color, oily smell like odor and homogenous in nature. The pH measured for formulations ranged from 5.52 to 5.94 (Table 1).


The encapsulation efficiency (EE) of 5-FU and curcumin are presented in Table 1. The percentage of encapsulation efficiency of the 5-FU ranged from 70.58 – 87.13%, where X2 exhibited the high-est and curcumin oscillated between 74.10 – 91.20%, where the highest was for B2.

Particle size

Particles in formulations above 25:75 (5-FU:curcumin) were higher compared to other for-mulations.

For formulations with 60% surfactant, the aver-age particle size was in the range of 15.68 – 26.35 nm while for formulations with 40% surfactant was between 11.27- 21.35 nm. The polydispersity index (PDI) values for the LC formulations were between 0.1 - 0.4 indicating that most of the vesi-cles ranged from homogenous to heterogeneous in particle size (Jukanti et al., 2011). In this study, four formulations with 50:50 ratio of drugs were shown to have PDI values between 0.1 - 0.2. The zeta potential ranges between -18 to -28 mV for all the formulations.




Table 1. Formulation composition and physiochemical properties of liquid crystal formulation.

Formulation

Code

Drug: Tween 80 (%)

5-FU: Curcumin pH Encapsulation efficiency

(%) ± SD

Vesicle size

(nm) ± SD Polydispersity index

Zeta potential

(mV)

5- FU Curcumin

A1 0.3:40 25:75 5.59 72.05 ± 2.84* 74.10 ± 2.09** 14.67 ± 0.58* 0.132 ± 0.05 -22.3 ± 1.02

A2 0.3:40 50:50 5.61 84.23 ± 3.64* 86.93 ± 1.37* 21.35 ± .056* 0.121 ± 0.02 - 26.2 ± 2.47

A3 0.3:40 75:25 5.54 79.10 ± 2.71* 80.39 ± 1.33* 20.24 ± 1.38* 0.424 ± 0.14 - 19.2 ± 1.14

B1 0.5:40 25:75 5.52 74.12 ± 1.25* 76.94 ± 2.45* 11.27 ± 1.27* 0.402 ± 0.15 - 18.1 ± 1.04

B2 0.5:40 50:50 5.53 86.35 ± 1.67* 91.20 ± 1.84* 16.35 ± 2.30* 0.236 ± 0.04 - 23.2 ± 1.74

B3 0.5:40 75:25 5.50 80.23 ± 1.57* 84.45 ± 2.93* 20.23 ± 2.64* 0.415 ± 0.11 - 17.4 ± 1.05

X1 0.3:40 25:75 5.85 70.58 ± 2.69* 74.45 ± 1.74** 11.36 ± 0.67* 0.187 ± 0.09 -23.5 ± 1.02

X2 0.3:60 50:50 5.67 87.13 ± 2.54* 89.48 ± 3.91* 22.67 ± 1.05 0.130 ± 0.07 - 28.3 ± 1.07

X3 0.3:60 75:25 5.68 76.69 ± 1.10* 79.83 ± 2.98* 25.67 ± 1.89* 0.408 ± 0.10 - 20.5 ± 1.14

Y1 0.5:60 25:75 5.52 73.15 ± 2.34* 75.74 ± 1.03** 15.68 ± 0.23** 0.428 ± 0.12 - 23.5 ± 1.16

Y2 0.5:60 50:50 5.94 83.27 ± 2.02* 86.05 ± 1.66* 21.67 ± 1.89* 0.204 ± 0.04 - 28.8 ± 1.83

Y3 0.5:60 75:25 5.84 79.07 ± 2.56** 81.74 ± 1.83* 26.35 ± 0.75* 0.406 ± 0.10 - 24.8 ± 1.32

Ratio for water: glycerin for all the formulations were 7:1; All formulations are Light-yellow, oily smell and homogenous in nature. The results are presented as mean ± SD. *p <0.01 and **p<0.001 indicate significant differences with respect to baseline valor of their respective group or among groups as shows the column at right. The one-way analysis of variance test followed by Bonferroni's multiple comparison between the formulations.




Table 2. The percentage of cytotoxicity of different surfactants on HeLa cells.

Concentration (µg/mL)

Percentage of cytotoxicity

Tween 80 Labrasol Cremophor

0.75 0.02 ± 0.001* 0.04 ± 0.003* 0.5 ± 0.001*

1.50 0.04 ± 0.002* 0.02 ± 0.002* 0.6 ± 0.002*

3.00 0.04 ± 0.001** 0.06 ± 0.001* 0.9 ± 0.001**

6.25 0.04 ± 0.002* 0.08 ± 0.002* 5.7 ± 0.4*

12.50 0.04 ± 0.001* 0.09 ± 0.002** 8.2 ± 0.06*

25.00 0.04 ± 0.002* 1.6 ± 0.3* 30.8 ± 1.05*

50.00 0.04 ± 0.003* 1.8 ± 0.2* 45.5 ± 1.3*

100.00 0.04 ± 0.001* 2.4 ± 0.3* 82.5 ± 1.8*

The results are presented as mean ± SD. *p <0.01 and **p<0.001 indicate significant differences with respect to baseline valor of their respective surfactant. The one-way analysis of variance test followed by Bonferroni's multiple comparison between the formulations.

Environmental Scanning Electron Microscopy (eSEM)

Based on the eSEM results, the majority of the particles were in the size of 5 µm. The particle siz-es from eSEM and from zetasizer were different due to the nature of the method used. It was ob-served that there were spherical structure for-mations of micelles in each formulation. The re-sults have been shown in the Fig. 1A-D. Fig1C and 1D showed dark spots in the vesicles.

Fourier transforms infrared spectroscopy (FTIR)

FTIR spectra of the drug with combinations of Tween 80 were obtained.

Both 5-FU and curcumin showed its characteris-tic peaks of bands at 3425.69 cm-1 – 3028.31 cm-1 and 1500.67 cm-1. Also, an additional band at 1431.23 was observed for curcumin. The bands at 1658.84 cm-1 and 1222.91 cm-1 are attributed by to (C=O) and δ (N-H) of 5-FU, respectively (Wu et al., 2015) (Fig. 2). (Derenne et al., 2013). The band at 1431.23 cm-1 is attributed to C=C aromatic while the band at 1500.67 cm-1 is for C=C olefinic of cur-cumin (Rajabalaya et al., 2015). The bands at 1658.84 cm-1 and 1222.91 cm-1 are attributed to

C=O and δ (N-H) of 5-FU, respectively (Wu et al., 2015) (Fig. 2).

Dissolution

The graphs in the Fig. 3A-B, depicted that the percentage of drug dissolution of 5-FU, was higher (A2: 40.23%) at PBS pH compared to VSF pH (A2: 36.23%). In the Fig. 4A-B, the highest percentages of drug dissolution of 5-FU were at pH 7.4 (PBS: 56.36%) and pH 4 (VSF: 49.01%), respectively. The percentage of dissolution of 5-FU at pH 7(PBS) A2: 40.23 > B2: 47.39 > X2: 50.36 > Y2: 56.36 compared to pH 4 (VSF) A2: 36.23 > B2: 43.64 > X2: 45.67 > Y2: 49.01. The percentages of dissolution of cur-cumin of different formulations were at pH 7 (PBS) A2: 44.85 > B2: 51.36 > X2: 58.61 > Y2: 64.38 com-pared to pH 4 (VSF) A2: 41.03 > B2: 49.37 > X2: 57.85 > Y2: 68.75 (Fig. 3C-D).

Drug efficacy study

Determination of half maximal inhibitory con-centration (IC50) of the combination of curcumin and 5-FU on HeLa cells was determined. From the Fig. 4, it could be observed that the IC50 for curcu-min and drug combination was 22.8 and 40.0 µg/mL respectively, while the IC50 for 5-FU was higher, thus it is not stated in Fig. 4.




A B

C D

Figure 1. ESEM figures of (A) blank liquid crystal, (B) formulation A2, (C) formulation X2 and (D) formulation Y2.

DISCUSSION

Physiochemical properties

The formulations prepared need to follow the desired criteria where it needs to have gel-like ap-pearance and do not contribute to any toxic effects to the cells. LC prepared with both Labrasol and Cremophor do not follow the desired criteria, namely pH and toxicity profile. So, they were not used in further studies. This finding was support-ed by previous studies, where it was reported that

Cremophor EL increased the toxicity and lead to hypersensitivity reactions in certain individuals (Zhao et al., 2007). Therefore, Tween 80 with ap-propriate properties for vaginal drug delivery was selected and used for further studies.

The LC formulations contained inadequate amounts of aqueous phase; therefore, the pH val-ues were determined for quality control and to evaluate the compatibility with vaginal pH. All the LC gel formulations were within the range of normal vaginal pH (Lucero et al., 1994). The ob-




tained pHs were suitable to be used in vaginal application (Musa et al., 2017).

LC prepared with 40 and 60% of Tween 80 were chosen as final formulations for subsequent stud-ies accredited to their physical properties. In addi-tion, Tween 80 has also been reported to have been used in the preparation of nanoparticles and nanoemulsions (de Mattos et al., 2015; Bonferoni et al. 2019) ascertaining the utility of the surfactant to be safe in various formulations.


EE controls the important positive relationship with oil to the concentrations of surfactants and water. The encapsulation of 5-FU and curcumin is primarily due to its capability to liquify in the mol-ten oil phase and its partition between the hydro-philic phase and surfactant (Das et al., 2018). All the LC formulations showing higher values of the

EE, may be due to lesser bilayer permeability as well as higher lipophilic bilayer, leading to effec-tive interpolation of the lipophilic drugs inside the core of the hydrophobic bilayers (El-Samaligy et al., 2006). However, the EE values do not signifi-cantly increase with increasing amounts of surfac-tant or drug contents. The higher amount of drugs and Tween 80 may cause the hydrophilic phase viscosity to increase, which then leads to the con-trolled drug release from the vesicles (David et al. 2018). It is postulated that lower quantity of aque-ous phase leads to higher viscosity of LC formula-tion vesicles with bilayer formation ushering in accommodation of higher amount of drug in the surfactant hydrophobic chains (El-Samaligy et al., 2006; Rajabalaya et al., 2016). This would aid in the formation of suitable viscosity and phase of LC gels phase, subsequently supporting the delivery of the drug in a controlled release manner.

Figure 2. Stacked spectrum of the 5-FU alone, curcumin alone and combination of 5-FU and curcumin.

Purple: 5-FU Red: CMN Blue: combined




A B

C D

Figure 3. (A) Dissolution profile of 5-FU with PBS; (B) dissolution profile of 5-FU VSF; (C) dissolution profile curcumin with PBS; (D) dissolution profile curcumin with VSF.

Particle size

The vesicle size distributions were homogene-ous and were within the acceptable limits for Tween 80 based formulations. The zeta potential of the polysorbate LC formulations with higher nega-tive values incline to resist vesicular aggregation consequently does not produce particle-particle aggregation (Scholes et al., 1999; Zhang et al., 2010). This phenomenon may be due to the low molecular weight of Tween 80 leading to low zeta

potential values. This is evidenced by the lesser particle size of Tween 80 than higher ratio formu-lations (Rajabalaya et al., 2016). The particle size is influenced by the synthetic drug, 5-FU, When FU ratio increases the particle also increases, which may be due to the lipophilic nature of the drug. The size of the vesicle and its distribution in the formulations are of paramount importance for the combination drugs which determines the drug delivery profile of the formulation (Plessis et al., 1994).




Combination Index (CI) at specific Effective Dose (ED)

Combination ED50 ED75 ED90 ED95

5-FU and Curcumin 0.64804* 0.72285* 0.81759** 0.88973*

Figure 4. Graph of % toxicity vs. concentration (µg/mL).

The results are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences with respect to base valor. 5-FU: 5-fluoracyl; CMN: curcumin; Combi: Combination of 5-FU + CMN.

Environmental Scanning Electron Microscopy (eSEM)

eSEM evidently demonstrated that the lower concentrations of Tween 80 containing LC formu-lations formed very small spherical shape with compared to higher ratios of components of 5-FU and curcumin. In the lower concentration of Tween 80-based formulations, the vesicles have lesser spherical shape compared to higher concen-trations formulations. The repulsion between the surfactant head groups lead to higher curvature in the micelles, forming spherical vesicles (Bengt et al., 2014). The higher value of zeta potential ob-served in the polysorbate based gels indicating the better dispersion of LC vesicles in the formulations when diluted with the distilled water (Shustova et al., 2011). It was observed that gels with lower concentration of drug produced spherical micellar

structures. Moreover, micelles with higher concen-tration of the drug content showed black dotted on the surface that indicates the distribution of hy-drophobic drug, either 5-FU or curcumin solubil-ized in hydrophobic chain molecules. Thus, this postulates that higher amount of drug is present in the micelles.

In eSEM, the prepared sample is placed directly on the sample holder, where the sample is evapo-rated resulting in the aggregation of the sample thus making the size bigger. While in zetasizer, the sample is diluted, thus leading to smaller particle size. Although the particles form micelles, it is not confirmed whether the micelles are normal or re-versed phase micelles. Thus, a more powerful mi-croscope such as cryo-Transmission Electron Mi-croscopy (cryo-TEM) need to be use in order to observe the micelles at higher magnification to differentiate the micelle type.




Fourier transforms infrared spectroscopy (FTIR)

Based on the stacked spectrum, all bands from 5-FU alone and curcumin alone were present in the spectrum of the combination of the drugs (Fig. 2). This indicates that there was no chemical inter-action between the chemicals when mixed togeth-er. The individual components curcumin, 5- FU and Tween 80 combination mixtures demonstrated compatibility of the surfactants with the drug in the formulation as the spectra was without signifi-cant peak shifts. This indicates that no chemical interaction between the drug and the surfactant. There were few characteristic peaks of the drug, which were overlapping in the region as that of the surfactants possibly due to the encapsulation of the drug between the layers.

Dissolution

The PBS with pH of 7.0 – 7.4 represents the vag-inal pH during menopause, and vaginal simulated fluid (VSF) with pH of 3.5 – 4.9 represents vaginal pH during adult life. Dissolution study was car-ried out in both of these simulated fluids to have wide range of applications for the liquid crystal gel formulation.

LC formulations showed that they were a good delivery vehicle throughout this research. They were suitable to be used as topical drug delivery system since they have mucoadhesive properties, which increase the absorption of drugs when ap-plied topically and were able to incorporate both 5-FU and curcumin. The consistent drug release demonstrates the stability of both the drugs used. Furthermore, in the dissolution study, the drug release increases with time indicating sustained drug release profile and good percentage of drug dissolution (Ahn et al., 2010). It is also seen in both buffer systems, namely PBS and VSF, that the de-livery of the drug is increasing and did not reach plateau, which indicates that there is drug still entrapped in the liquid crystal system. This signi-fies that this LC gel system has a sustained release profile.

The dissolution behaviors show an almost simi-lar pattern with other drug and surfactants ratio; such as when the drug and surfactant concentra-

tion increase the percentages of dissolution also increases. It may be due to higher surfactant influ-encing drug dissolution, which may be due to im-proved physicochemical properties of drug like solubility, liquid crystalline forms, particle size, and diffusivity of the compound (Jamzad and Fassihi, 2006). Dissolution study using VSF with the same drug release pattern exhibits compatibil-ity of the LC system in both pH environments. The higher curcumin percentages of dissolution com-pared to 5-FU, may be due to two-fold factors; firstly, the ability of the surfactant to solubilize the curcumin efficiently in the dissolution medium and secondly, the optimal particle size enhancing dissolution in both the pH mediums. This shows that the formulation is suitable to be used in both post-menopause patients and adult patients (Lara et al., 2005).

Drug efficacy study

The drug efficacy study indicates that 5-FU when given alone requires a higher concentration to produce its effects. Based on Chou (2006), the combination index (CI) value was 0.64804 and the CI value were in the range of 0.3 – 0.7 and showed synergism between 5-FU and curcumin. Another study showed that the CI value for both ED75 and ED90 were in the range of 0.7 – 0.85, which indicate moderate synergism while CI value for ED95 showed slight synergism between the two drugs (Liu et al., 2017). Another study by Du et al. (2006) only focused on the synergism of 5-FU and cur-cumin.

The current drug efficacy study shows syner-gism at ED50, for the combination of 5-FU and cur-cumin. The combination of the drugs is safe to be applied and with further study, the dose of 5-FU may be reduced while increasing the dose of cur-cumin. With this, the therapeutic effect may be maintained, while the side effects of 5-FU may be reduced (Seong-Ho A, 2010).

CONCLUSIONS

The present study demonstrates that the com-bination of 5-FU and curcumin in liquid crystal system is stable with appropriate pH that is suita-




ble for both adult female and post-menopause female. It also has suitable physicochemical prop-erties that fit the criteria to be used topically in the vagina. The sustained drug release profile in the dissolution study shows that the LC system re-leases the required amount of drugs for a very long time. The combination of 5-FU and curcumin is suitable to be used on cervical cancer HeLa cell line.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

The authors sincerely thank the School of Pharmacy, In-ternational Medical University, Kuala Lumpur, Malaysia for providing grant and lab facilities for the research work [BPharm B01/09-Res (17)2012].

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AUTHOR CONTRIBUTION:

Contribution David SR Refai SA Yain KR Mai C-W Das SK Rajabalaya R

Concepts or ideas x x x

Design x x

Definition of intellectual content x x

Literature search x x

Experimental studies x x x x x x

Data acquisition x x x x x

Data analysis x x x x

Statistical analysis x x x

Manuscript preparation x x x

Manuscript editing x

Manuscript review x x x x x x

Citation Format: David SR, Refai SA, Yain KR, Mai C-W, Das SK, Rajabalaya R (2019) Development and evaluation of liquid crystal systems of combination of 5-fluorouracil and curcumin for cervical cancer cell line. J Pharm Pharmacogn Res 7(6): 441–453.


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